Hidekazu Hata

Toxoplasma gondii Hsp70 as a danger signal in Toxoplasma gondii-infected mice

Abstract Toxoplasma gondii Hsp70, T gondii Hsp30/bag1, and surface antigen 1 messenger RNAs were shown to be useful in analyzing stage conversion of T gondii between bradyzoites and tachyzoites. The high-level expression of T gondii Hsp70 was correlated with mortality in interferon-γ knockout mice infected with T gondii. T gondii Hsp70 inhibited the induction of nitric oxide release by peritoneal macrophages of T gondii-infected mice. These findings identify T gondii Hsp70 as a danger signal during lethal, acute T gondii infection.

Arginine‐dependent generation of reactive nitrogen intermediates is instrumental in the in vitro killing of protoscoleces of Echinococcus multilocularis by activated macrophages

The interaction between protoscoleces of Echinococcus multilocularis and activated murine macrophages was examined in this study. Marked protoscolicidal activity was displayed by peritoneal macrophages (PM) activated with interferon‐γ (IFN‐γ), or IFN‐y plus lipopolysaccharide. Pretreatment of the parasites with heat‐inactivated specific murine infection serum, but not with normal serum rendered them more susceptible to PM killing. NG‐monomethyl‐Larginine, a competitive inhibitor of L‐arginine completely inhibited the killing activity of activated PM. while reconstitution of arginine‐free medium with L‐arginine restored the killing properties of the activated PM. The results show that activated PM have the ability to kill E. multilocularis protoscoleces in vitro and suggest that reactive nitrogen intermediates (RNl) play an important role in the mechanism. An oxygen‐mediated mechanism did not appear to play a role because scavengers of reactive oxygen species did not reduce the killing activity. The arginine‐dependent killing mechanism was enhanced by superoxide dismutase (SOD), probably because SOD might prolong the effect of nitric oxide. Secretion of RNI by activated macrophages may be capable of a significant role in preventing of the dissemination of E. multilocularis infection in vivo.

Anti-HSP70 Autoantibody Formation by B-1 Cells in Toxoplasma gondii-Infected Mice

ABSTRACT Formation of anti-Toxoplasma gondii HSP70 (TgHSP70) antibody cross-reactive to mouse HSP70 (mHSP70) was observed in the sera of BALB/c (a resistant strain) and C57BL/6 (B6; a susceptible strain) mice after peroral infection with T. gondii cysts of the Fukaya strain. The levels of anti-mHSP70 immunoglobulin G (IgG) autoantibody production in B6 mice were higher than those in BALB/c mice. The isotype and subclass of IgG of anti-TgHSP70 monoclonal antibodies cross-reactive to mHSP70 were μ and γ3. Anti-mHSP70 autoantibody in T. gondii-infected BALB/c and B6 mice was shown to be produced by the CD5+subset of B cells (B-1a cells) but not by conventional B cells (B-2 cells). The epitopes recognized by anti-mHSP70 autoantibody were located primarily in the C-terminal fragment of mHSP70.

Organ Infectivity of Toxoplasma gondii in Interferon-γ Knockout Mice

To determine the influence of interferon (IFN)-γ on the organ infectivity and on the genetic susceptibility of susceptible (C57BL/6) and resistant (BALB/c) strains after peroral infection with cysts of Toxoplasma gondii, IFN-γ knockout (KO) mice in C57BL/6 and BALB/c backgrounds were utilized. The kinetics of the changes in T. gondii abundance were evaluated with a quantitative competitive polymerase chain reaction assay in various organs at different times after peroral infection. In IFN-γ KO mice, a T. gondii-specific gene, SAG1, was detected in all organs examined, and the protozoan proliferated much more actively than in wild-type mice. The abundance of T. gondii was much higher in mesenteric lymph nodes and the heart than in other organs. In contrast, in the nervous system organs and kidneys, only a weakly detectable reaction was observed. Toxoplasma gondii grew at a more rapid rate in the organs of IFN-γ KO C57BL/6 mice than in the organs of IFN-γ KO BALB/c mice during the course of infection. Destruction of the IFN-γ gene showed remarkable effects on the infectivity in both susceptible and resistant mice.

Protective immunity induced by vaccination with SAG1 gene-transfected cells against Toxoplasma gondii-infection in mice

To develop a vaccine by augmenting the protective cellular immunity against Toxoplasma gondii (T. gondii)‐infection, T. gondii SAG1 gene‐transfectants were established by using RMA.S (H‐2b), a murine transporter associated with the antigen processing (TAP) molecule‐deficient lymphoma line, as a host antigen‐presenting cell (APC). Immunization of C57BL/6 mice with the SAG1‐transfected RMA.S induced CD8+ cytotoxic T lymphocytes (CTL) specific for not only SAG1‐transfected RMA.S but also T. gondii‐infected RMA.S, and elicited protective responses to infection with a virulent T. gondii strain, RH.

Toxoplasma gondii: difference of invasion into tissue of digestive organs between susceptible and resistant strain and influence of IFN-gamma in mice inoculated with the cysts perorally.

Because it is widely accepted that there is a significant difference in susceptibility to chronic infection by Toxoplasma gondii among inbred mouse strains with different genetic backgrounds, we compared the distribution of the protozoa in digestive organs at early stages of infection between resistant (BALB/c) and susceptible (C57BL/ 6) mice after peroral infection with Fukaya strain cysts. Furthermore, to determine the influence of interferon gamma (IFN-gamma) on the infectivity of the cysts to the digestive tract, homozygous IFN-gamma knockout mice were utilized. Quantitative competitive polymerase chain reaction (QC-PCR) was employed to assess the distribution of T. gondii in different organs at various times after ingestion of cysts. SAG1, a T. gondii-specific gene, was detected in the small intestine and the caecum in wild-type C57BL/6 mice and in the whole digestive tract in IFN-gamma knockout C57BL/6 at 24 hr after infection. No detectable reaction in QC-PCR was observed in BALB/c mice at 24 hr after ingestion of the cysts. Destruction of the IFN-gamma gene showed less effect on the resistance to infection in BALB/c mice, but remarkable augmentation of infectivity of T. gondii to the rectum and peripheral blood was observed in C57BL/6 mice.

Establishment of gene-vaccinated skin grafting against Toxoplasma gondii infection in mice.

Vaccine effects of in vivo gene-vaccinated skin graft were evaluated against Toxoplasma gondii (T. gondii) infection. By using a gene gun, cDNA coding T. gondii SAG1 molecule was intracutaneously vaccinated into C57BL/6 (B6; a susceptible strain), BALB/c (a resistant strain) and (C57BL/6 x BALB/c) F1 (CBF1) mice, and the gene-vaccinated skin of these strains was transplanted to CBF1 mice. Regarding the antibody production against SAG1, CBF1-recipient mice transplanted with the SAG1 gene-vaccinated B6 skin were high responders, whereas CBF1 mice skin grafted with vaccinated skin of both BALB/c and CBF1 mice were low responders. The donor-derived LC/DC migrated to the draining lymph nodes of the recipients from the skin graft within 3 days. The vaccine effect against T. gondii challenge infection was obtained in CBF1 mice which received the skin graft of the SAG1 gene-vaccinated BALB/c mice.

ESSENTIAL AMINO ACIDS AND OTHER ESSENTIAL COMPONENTS FOR DEVELOPMENT OF ANGIOSTRONGYLUS COSTARICENSIS FROM THIRD-STAGE LARVAE TO YOUNG ADULTS

Third-stage larvae of Angiostrongylus costaricensis were cultured to the young adult stage in Waymouths chemically defined medium MB 752/1, which contained 18 amino acids, 11 vitamins, glutathione, hypoxanthine, and glucose in a balanced salt solution. Nutritional requirements were examined by deletion of single components from Waymouths medium. Ten amino acids, namely L-arginine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-threonine, L-tryptophan, and L-valine, were shown to be essential for the parasites development. L-Aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, glycine, L-proline, and L-tyrosine were not essential. Among the 11 vitamins, only choline chloride was essential for the development. The deletion of pyridoxine from the medium adversely affected parasite development. Glucose was also required by the worms, but glutathione and hypoxanthine were not required for their development. When the 10 essential L-amino acids were replaced individually by D-isomers of the same amino acids, none supported larval development in the manner of the L-amino acids.

Protective immunity to Schistosoma japonicum infection depends on the balance of T helper cytokine responses in mice vaccinated with γ-irradiated cercariae

Although the strain difference in protection of mice to Schistosoma mansoni infection has been described, limited information is available in the case of Schistosoma japonicum. In the present study, we compared the protective immunity to S. japonicum infection and cytokine production in various strains of mice vaccinated with γ‐irradiated cercariae. A significant reduction in worm recovery was observed in male and female mice of DBA/2 at a 6‐week interval between vaccination and a challenge infection, whereas vaccinated mice of C57BL/6, C57BL/10, (C57BL/6 × DBA/2) F1 (B6D2F1) and (C57BL/10 × DBA/2) F1 (B10D2F1) showed no detectable level of protection. No sex‐linked difference in development of resistance was observed in any of the strains so far examined. Vaccination with γ‐irradiated cercariae twice with a 3‐week interval also induced significant protection against a challenge infection in DBA/2 but not in BALB/c or C57BL/6 strains. Further studies demonstrated that spleen cells of vaccinated C57BL/6 mice produced lower levels of IFN‐γ compared to the cells of vaccinated BALB/c and DBA/2. On the other hand, production of IL‐10 by spleen cells was relatively higher in BALB/c mice than in the other two strains. Macrophages that had been stimulated with spleen cell culture supernatants derived from vaccinated DBA/2 damaged schistosomula more effectively than cells stimulated with supernatants derived from the other strains. These results suggest that different levels of protection observed among strains of mice depend on the balance of cytokine responses which consequently activate or suppress macrophage‐mediated damage to schistosomula.

Effects of recombinant tumour necrosis factor on antibody‐dependent eosinophil‐mediated damage to Schistosoma japonicum larvae

Summary Human recombinant tumour necrosis factor (rTNF) enhanced monoclonal IgE‐dependent eosinophil‐mediated cytotoxicity to schistosomula of Schistosoma japonicum in a dose‐dependent manner. The enhancing effect of rTNF was also observed for the antibody‐dependent cytotoxicity of a human eosinophilic leukemia cell line, EoL‐3, but not of another cell line, EoL‐1. Observation by a slow‐motion movie camera demonstrated that activated EoL‐3 cells adhered to the surface of schistosomula by 6 h after incubation, which triggered intracellular movement of eosinophil granules. The granules were concentrated toward the surface of the larvae and then degranulation started. The cell membrane was left as a balloon‐like remnant. Cell sorting analysis by FACStar indicated that the expression of receptors for C3bi (CR3) and low affinity FcR for IgE (FceRII) increased on the surface of EoL‐3 cells after stimulation with rTNF, while this was not observed for EoL‐1 cells.